Using blood culture platforms for commercial sterility tests

ABSTRACT

A system that indicates the presence or absence of microorganisms in fluid food products. The system has a bottle for receiving sample to be tested. The bottle has a sensor that will monitor and detect changes in at least one sample parameter, but no additives that contain nutrients that support microbial growth. The bottle is placed in an incubator and the sensor in the bottle is monitored for changes. The incubator is programed so that, if the sensor detects that the value of the monitored parameter has reached a certain value, then the sample is determined to be positive for microbial growth.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a national phase entry under 35 U.S.C. § 371of International Application No. PCT/US2015/032445 filed May 26, 2015,published in English, which claims priority from Chinese PatentApplication No. 201410227620.9, filed May 27, 2014, all of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Food borne illnesses are a matter of public concern. Most developednations have policies and procedures in place to ensure a reliably safefood supply, free of contamination by pathogens that can cause foodborne illness.

Many countries have developed tests and protocols for inspecting foodfor contamination to ensure that contaminated food does not enter thefood supply. One example of such a protocol is the National Food SafetyStandard of the People's Republic of China. In the United States testsand standards for monitoring the food supply for pathogens arepromulgated and enforced by the United States Department of Agriculture.

The goal of these tests is to keep unsafe or potentially unsafe foodfrom consumers. However, as with any such test, the integrity of thetest is crucial to identify foods that are potentially unsafe forconsumption and to keep those from consumers without having samples testfalsely for pathogens or contamination. False positives are an economicburden to society, both suppliers and consumers alike. So any method,system or device that inspects food must be accurate in itsidentification of foods that present a real public health risk.

The protocol for the Chinese inspection standard is illustrated inFIG. 1. Basically, sample 100 (such as, for example, milk) while stillin the container in which it is packaged, is examined (120) for signs ofcontainer damage or a breach in container integrity. If the container isdetermined to be sound, the container is placed in an incubator at step140. The container is stored in the incubator at a temperature of about35° C. for 10 days. After that time, the container is inspected visuallyfor any signs of bloating or expansion that are indicative of thepresence of pathogens. If the container bears signatures of microbialgrowth in the container during incubation, the container is cooled in arefrigerator before the sample is opened and inspected. This ensuresthat the contaminated sample will not aspirate out of the container whenit is opened. Control samples are also placed in the refrigerator as acontrol for the incubated samples. When the pH of the contents of theincubated samples is measured, it is compared with the pH of thecontents of the refrigerated samples. A pH difference of 0.5 or more isan indication of microbial growth in the incubated sample.

Upon incubation (and cooling when appropriate) containers that exhibitphysical signs of microbial growth are opened (150). An aliquot of thecontents is removed (160) and placed in a sterile container. Thereserved sample is inoculated onto culture media and the sample iscultured to identify the microbes that are the source of the microbialcontamination.

When the container is opened, the pH of the contents is measured and theorganoleptic properties of the sample (e.g. the properties experiencedby the senses such as smell, color, etc.) are inspected (170). Thesample is then prepared for microscopic examination (180). Themicroscopic examination is intended to identify the source of themicrobial contamination and to determine if the microbes are pathogenic.After inspection a report is issued. The report indicates that thesample is either acceptable (i.e. commercial sterilization) or notacceptable (non-commercial sterilization).

The methods described in FIG. 1 are time consuming due to the long waitsfor incubation of the sample. Attempts have been made to accelerate theinspection of such samples using an automated system for detectingmicrobial growth in samples. Zheng, J., et al., “Study on rapid detectcommercial sterilization of fungus (i.e. mushroom) cans with BacT/ALERT3D system,” Food Science and Technology, No. 9, pp. 196-199 (2007)report three days to detection using the BacT/ALERT 3D system. InBacT/Alert the sample is placed in a bottle with culture media. Thebottle also has a CO₂ sensor. The sensor detects the presence of carbondioxide in the sample. If the system detects an increase in the carbondioxide content of the sample bottle beyond a certain level duringincubation, the system flags the sample as positive for microbialgrowth. For this study, two kinds of bottles containing media were used.One (the i AST bottle) contained media for aerobic bacteria detectionand the other (the i NST bottle) contained media for anaerobic bacteriadetection. The bottles (which contained media) were spiked with lowlevels of different kinds of bacteria and 10 ml of the product (solutionin mushroom cans). Time to result using BacT/Alert was listed in Table 1and was reported as in the range of 16 hours to about 30 hours. Alsoreported is a non-spiked sample experiment using 45 cans containingmushrooms. The results from the BacT/Alert were compared with theresults from inspection using the Chinese protocol referred to as thestandard test protocol herein. BacT/Alert identified one positive sampleamong the 45 samples, and the contamination was verified using thestandard test protocol.

Zheng, J., et al., “Application of BacT/Alert 3D System in detection ofCommercial Sterilization of Konjac Cans,” Food Science, Vol. 29, No. 10,pp. 463-467 (2008) describes testing Konjac Cans with BacT/Alert 3D.Samples from fifty-nine cans (not spiked) were tested. The results werecompared with the results of cans tested using the standard test. Threecontainers of sample were used for this test. From one container, 10 mlof sample were added to each BacTAlert bottle. Specifically, 10 ml ofsample was added to each of the i. AST and ii. NST bottles. The secondcontainer was analyzed by the standard test protocol and the thirdcontainer was held at room temperature for follow up tests. For the canstested using the standard test protocol, none of the cans failed qualitycontrol. However, nineteen of the samples tested with BacT/Alert werereported as positive for microbial growth in one or both bottles. Thesepositive results were considered false positives when compared with thecontrol (testing by the standard test). Among the nineteen BacT/Alertpositive samples, three of them were confirmed not to containmicroorganisms. To confirm the presence or absence of microorganisms,sample from positive bottles were inoculated on five different standardmedia to detect the present or absence of microbial growth. Meanwhilesample from the positive bottles was also examined under a microscopefor evidence of microbial growth. From these it was determined that thethree positive samples were false positive samples since, for all threeof these samples, there was no sign of microbial growth from either theinoculated cultures or the samples subjected to examination bymicroscope. Various bacteria were isolated from the other sixteenbottles identified as positive through testing using BacT/Alert.According to this article, the Konjac cans probably contain some livemicroorganisms, but those microorganisms cannot grow in the Konjac cansdue to the high pH environment (10-12.5). Therefore, in these cans,whatever microorganisms might be present are not pathogenic, did notrender the contents unsafe for consumption, and would have passed thestandard testing protocol established by the Chinese government.Nevertheless, once the samples were diluted in bottles containingculture media for testing in BacT/Alert, the microorganisms were able togrow and triggered the BacT/Alert instrument to report positive results.

Dong, R., et al. Heilongjiang Province CDC, Chinese Primary Health Care,Vol. 23, No. 12 (December 2009) describes the use of BacT/Alert forevaluating milk sterility. In this study, one kind of ultra-hightemperature (UHT) milk was spiked with 2 bacteria strains (E. coli andB. cereus). As both bacteria strains grow in an aerobic environment,only the AST bottle was evaluated. The test demonstrated that a largervolume of sample was more sensitive, but did not address the issue offalse positives as it did not compare the results obtained usingBacT/Alert with the standard test protocol.

Accordingly, alternative methods and systems for testing food forpathogens that have reduced time to detection but compare favorably withstandard test protocols in terms of the number of false positives orfalse negatives continue to be sought.

BRIEF SUMMARY OF THE INVENTION

A system for testing fluid foods for contamination. The system uses asample container that is adapted to be used in an incubator thatmonitors a sample disposed in the container for evidence of microbialgrowth. In this regard there is a sensor disposed in the container. Thesensor monitors at least one parameter of the sample as the sample isheated in the incubator. The parameter is a condition of the sample thatwill change should microbial growth occur in the sample duringincubation. In this regard the sensor will provide a response to changesin a sample parameter that change in response to the metabolic activityof microorganisms. Such parameters include the concentration of oxygenin the sample, the concentration of carbon dioxide in the sample, or thepH of the sample.

When monitoring the sensor, the system will flag a bottle as positivefor the presence of microorganisms if the measured value of theparameter exceeds a predetermined value. The sensor is placed in thecontainer such that, when the sample is introduced into the container,the sensor is in contact with the sample. The system is programmed withthe predetermined threshold value of the monitored parameter associatedwith microbial growth. In this regard, the measured parameter willincrease if the measured parameter is produced by microorganismmetabolic activity (e.g. CO₂). It follows that the parameter willdecrease if the value measure parameter is consumed by microorganismmetabolic activity (e.g. O₂).

The system has a receptacle in the incubator for receiving the samplecontainers. The sample containers are positioned such that the systemdetector can monitor the sensor during incubation of the sample in theincubator. The sample containers are presented for the introduction ofsample therein without containing additives that include nutrients formicrobial growth.

Also described is a method for testing fluid foods for microbialcontamination. In this method a test sample is drawn from a commerciallypackaged sample under inspection. The test sample is introduced into asterile container with a sensor that monitors a parameter associatedwith microbial growth. The sample is introduced into the container inliquid form. However, the sample can be a liquid sample (for examplemilk) or brine or liquid packaged with an otherwise solid sample, or asolid sample that has been liquefied for testing. The sample is referredto as a liquid sample herein, with the sample under test being liquid atambient and testing temperatures. The sensor is placed in the containerso that it will contact the test sample during subsequent incubation.The container has no nutrients that support microbial growth therein.The sample is then incubated at a temperature of about 30° C. to 38° C.while monitoring the sensor for changes in the parameter monitored bythe sensor. If the sensor reading is a predetermined value associatedwith microbial growth, the sample is flagged as a sample as positive formicrobial growth.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, advantages and novel features of the inventionwill be more readily appreciated from the following detailed descriptionwhen read in conjunction with the accompanying drawings, in which:

FIG. 1 is a flow diagram of the standard test protocol;

FIG. 2 is a block diagram of a system employing multiple incubation andmeasurement instruments according to an embodiment of the presentinvention, which each uses photothermal spectroscopy to monitor theconcentration of a gas, such as oxygen or carbon dioxide, in samplebottles, to thus detect for microorganism growth in the sample bottles;

FIG. 3 is a detailed view of an instrument employed in the system shownin FIG. 1;

FIG. 4 is a cutaway top view of the instrument in FIG. 3; and

FIG. 5 is a BACTEC bottle configured for use in the system and methoddescribed herein.

DETAILED DESCRIPTION

A system 100 for detecting growth of microorganisms in sample culturesaccording to an embodiment of the present invention is shown in FIG. 2.As illustrated, the system 100 includes a plurality of incubation andmeasurement modules 102 that are connected to a central computer 104.The central computer 104 can control the incubation temperatures andtimes, as well as the timing of the measurements performed by themodules 102 and can collect and classify the data readings obtained bythe modules 102. The system 100 can also include a data output device,such as a printer 106 that can be controlled by the central computer 104to print data readings obtained by the incubation and measurementsmodules 102.

Examples of such systems are well known to those skilled in the art andare not described in detail herein. One example of such a system is theBD BACTEC™ FX40 which is commercially obtained from Becton Dickinson.The operation of the BD BACTEC™ FX40 is described in the BD BACTEC™ FX40Instrument User's Manual which is Document Number 8090414 and CatalogNumber 441980 which is incorporated by reference herein. The operationof BD BACTEC™ FX40 and other such instruments (for example Soleris® fromNeogen Corporation of Lansing Mich.) is well known to one skilled in theart and not described in detail herein.

Further details of the incubation and measurement modules 102 are shownin FIGS. 3 and 4. As illustrated, each incubation and measurement module102 in this example includes a housing 108 and two shelves 110 that canbe slid into and out of the housing 108 in a direction along arrow A.Each shelf 110 includes a plurality of openings 112, each of which isadapted to receive a sample bottle 114. The openings 112 are arranged ina plurality of rows and columns as shown, and each shelf 110 can haveany practical number of openings. For example, the openings 112 can bearranged in nine rows, with nine columns in each row, thus totaling 81openings 112 per shelf 110.

When a sample is to be analyzed by the incubation and measurement module102, the sample is placed in a sample bottle 114, and the sample bottle114 is loaded into a respective opening 112 in the incubation andmeasurement module 102. The sample bottle 114 is a closed sample bottlein this example. The incubation and measurement module 102 can furtherinclude a keyboard, a barcode reader, or any other suitable interfacethat enables a technician to enter information pertaining to the sampleinto a database stored in a memory in the incubation and measurementmodule 102, in the central computer 104, or both. The information caninclude, for example, patient information, sample type, the row andcolumn of the opening 112 into which the sample bottle 114 is beingloaded, and so on.

Each incubation and measurement module 102 further includes a movablemonitoring assembly 116 (including sensor head housing 118, verticalshaft 128 and horizontal shaft 130) which is capable of monitoring thecontents of a medium in the sample bottles 114 by monitoring signalsfrom a sensor 210 disposed inside the sample bottle 114. See FIG. 5.Such sensors monitor a parameter in the sample bottle 114 duringincubation. Such samples include oxygen levels, carbon dioxide levels,pH, etc. The sensors will detect a change in such conditions over time.Such sensors are well known to one skilled in the art and not describedin detail herein. The incubator is configured so that, when the value ofthe parameter being monitored crosses a certain threshold, the systemflags the bottle (and its contents) as positive for the growth ofmicroorganisms.

As noted above, it is important to ensure that the food supply is notcontaminated by pathogens. According to the world health organization(WHO/FAO), the goal for the food supply is commercial sterility. This isdefined as ‘the absence of microorganisms capable of growing in food atnormal non-refrigerated conditions at which the food is likely to beheld during manufacture, distribution and storage’.

The standard test protocol for commercial sterility that is recommendedby People's Republic of China National Standard for Food Safety (GB4789.26-2013), is to incubate the packaged food at 36° C.+/−1° C. for 10days, look for bloated packages, and then open all the packages for pHtesting, visual and microscopic inspections.

Although the cost per manual test is relatively low, the manual protocolis a time consuming and laborious process. The requirement for largecapacity incubators (to accommodate all manner of foods in bulkypackaging) adds significant cost to the food manufacturers and to thoseorganizations that test the food. In the embodiments described herein,conventional instruments for detecting microbial growth in bloodcultures have been adapted and modified to monitor food for the presenceor absence of microorganisms. The use of such instruments, such as BDBACTEC™ FX40, in the methods described herein provide a method andsystem that delivers much faster time to result while controlling thenumber of false positives and false negatives. Therefore, the system andmethods described herein provide a much faster time to result without asignificant increase in the number of false positives or false negativesachieved by prior methods.

In the method described herein, the food sample (e.g. milk) isintroduced into a sample bottle configured for use in a system thatmonitors blood cultures for microbial growth. The sample bottle 114 hasa sensor 210 disposed in the interior, preferably in a location wherethe sensor will be in contact with the sample when the sample isintroduced into the bottle. The bottle 114 is sterile and the air issubstantially completely evacuated therefrom.

The bottle volume is about 50 ml. The size of the bottle will vary, andso will bottle volume. However, bottles must be of a suitable volume toreceive a sample volume that will ensure adequate sensitivity of thesample. In this regard, if the bottles can only receive a very smallsample, then the sample might not be representative of the samplecontents in terms of the contaminants (if any) in the sample portion.For example, if the container size is one gallon, and the sample volumefor testing is only 10 ml, there is a real possibility that the sampleportion may not contain a microorganism even if they were present in thecontainer. For the method and apparatus described herein the minimumsample size would be no less than about 10 ml and preferably not lessthan 50 ml.

The bottle is preferably under some vacuum so that the sample can easilybe drawn into the sample container. Apparatus for drawing liquid samplesinto containers are well known and not described in detail herein.

The sensor is typically a CO₂ sensor. Examples of suitable sensors aredescribed in U.S. Pat. No. 5,998,517 to Gentle et al. which is herebyincorporated by reference. The sensor, e.g. a silicone sensor, isdisposed in a gel matrix. The sensor is interrogated during incubationto monitor changes in carbon dioxide levels in the sample. When thecarbon dioxide levels in the bottle exceed a predetermined threshold,the bottle is flagged as containing sample that has tested positive forthe presence of microorganisms therein.

The sample bottle 114 into which the sample is dispensed does notcontain any nutrient media that facilitates microbial growth. For thisapplication it is important to preserve the sample integrity and ensurethat nutrients for microbial growth come from the sample itself and notfrom additives to the sample. As illustrated in the following example,the number of false positive surprisingly increases when culture mediais present in the container in which the sample is disposed, incubated,and monitored for the presence or absence of microbial growth.

Using UHT milk as an example of a food sample, the sample bottles thatdid not contain culture media were prepared. In one embodiment thebottles are those that are configured for use in the BACTEC bloodculture device. The samples (50 ml) of UHT milk were obtained from threedifferent kinds of cartons. The samples were spiked with microorganisms(B. cereus, B. licheniform, C. perfringens, S. aureus; P. aeruginosa; L.fermentus). For comparison, 10 ml of the same spiked UHT milk sampleswere also added to BACTEC bottles containing Lytic/Anaerobic media. Allthe BACTEC bottles were loaded in a FX200 or BACTEC 9240 for detection.

UHT milk samples spiked with the same concentrations of microorganismswere tested by the standard test protocol described above. The resultsusing empty BACTEC bottles with 50 ml of milk samples were shown tobetter correlate to the results obtained when the standard test protocolwas used. Although applicants do not wish to be held to a particulartheory, applicant believe that the lower false negative rates forsamples tested in bottles without media compared to samples in bottleswith media are due to the larger sample size (50 ml vs 10 ml) and/or theselection of a media that yields a false result. A larger sample size ismore sensitive to samples that have a low concentration of organisms.

It was also observed that certain bacteria (e.g. C. perfringens) did notgrow in two kinds of milk cartons when those cartons were tested usingthe standard test protocol described above. When 50 ml samples weretested in bottles without media using BACTEC, the same observation wasmade. Thus, the method described herein yields results compatible withthe established test protocol. In contrast, when 10 ml samples wereintroduced into bottles that contained Lytic/Anaerobic media, thosesamples tested positive for microorganism growth. This is interpreted asa false positive. Therefore, the rate of false positives was lower usingBACTEC bottles without media that with media (using the standard testprotocol.

Milk that has been pasteurized using ultra high temperatures (UHT milk)was obtained in 3 different kinds of cartons: i) Prepack (soft plastic);ii) Tetra Pack (hard plastic); and Tetra Brik (hard cardboard box). Theproducts were purchased from local supermarkets in China.

Bacteria stock solutions were diluted to a very low concentration andthe same amount was spiked into 2 identical cartons of UHT milk (0.01cfu-0.5 cfu/ml were spiked into each carton). One of the spiked cartonswas sealed and put into a 36° C. incubator (the prior art referencemethod, also referred to as the standard protocol), and the other onewas used for studies on BACTEC bottles with and without media.

In the reference method, the cartons were examined daily forleakage/swelling, and were opened right away for further examination ifabnormalities in the cartons were observed. Other cartons that appearednormal were left in the incubator for about 10 days before being openedfor further tests. The tests were: i) pH; ii) smell; iii) inspection forprecipitates or aggregated material; and iv) plating the material on TSAand RCM agars and looking for colony growth after incubation at 36° C.The samples were identified as contaminated samples if any of the aboveparameters appeared abnormal.

A portion of the samples were disposed in bottles that contained thestandard Lytic/Anaerobic media for BACTEC. This media is commerciallyobtained from Becton Dickinson and is not described in detail herein.

For another set of bottles, the media was aspirated out of bottles, aswas any residual air. In those bottles, 50 ml of spiked UHT milk wasinjected.

For a third set of bottles, 5 ml saline was added to empty BACTECbottles which were then autoclaved. Air in those bottles was aspiratedout by a syringe to generate sufficient vacuum in the bottle to draw inthe sample. Then ml spiked milk was injected into the bottles, and 2duplicate bottles were used for each experiment. The bottles were loadedinto a BACTEC FX 200 or a BACTEC 9240 for detection. Samples wereconsidered commercially sterile if the samples were still negative afterfive days of incubation.

The same spiked sample carton was the source of sample for studies inbottles without media and for the studies in bottles with media. Eachcarton contained more than 200 ml milk and therefore there was anadequate amount of sample for both studies. The amount of spiked sampleinjected in each bottle containing Lytic/Anaerobic media was 10 ml. Twoduplicate bottles were used for each experiment. For the sample spikedwith P. aeruginosa, one bottle containing aerobic media was used inaddition to two bottles with anaerobic media therein. Samples wereconsidered commercially sterile if the samples were still negative after5 days of incubation.

TABLE 1 Media Effect on Result for Samples Spiked with P. aeruginosaStandard Lytic/ Lytic/ Aerobic Anaerobic Anaerobic bottle bottle bottleCat# 442260 (Cat# 442265) Cat# 442265 With media 35 cfu Without mediaWith media 10 ml/sample 36° C. P. 50 ml/sample 10 ml/sample Time toIncubation aeruginosa/ Time to Result Time to Result Result (standardbox (h/min) (h/min) (h/min) method) Tetra Brik 13:15 Negative 16:13 8days, no 250 ml/box (Bottle 1) (Bottle 1) swelling, 13:15 Negative milk(Bottle 2) (Bottle 2) spoiled Tetra Pack 14:54 Negative 16:53 8 days, no231 ml/box (Bottle 1) (Bottle 1) swelling, 15:14 Negative milk (Bottle2) (Bottle 2) spoiled Prepack 13:14 Negative 16:03 8 days, no 200 ml/box(Bottle 1) (Bottle 1) swelling, 13:24 Negative milk (Bottle 2) (Bottle2) spoiled

Results demonstrated that not all organisms will grow in all media. Asdemonstrated above, P. aeruginosa grew in aerobic media but did not growin an aerobic media. Obviously, if bottles without media are used, onedoes not have to select a specific media in which the organism willgrow. If bottles without media are used, there is no concern with mediaselection. The test results from samples in bottles without media werecompletely consistent with the results for samples in media in which thetarget organism grew (the aerobic media). The results for samplesdisposed in bottles without media had complete correspondence for theresult for samples incubated using the standard protocol.

TABLE 2 Effect of Sample Size on Results with Samples Spiked with L.fermentum Lytic/ Anaerobic bottle Saline bottle Cat# 442265 Withoutmedia With media 50 ml/sample 10 ml/sample Time to Result Time to Result(h/min) (h/min) 36° C. Incubation Tetra Brik 23:35 23:04 8 days,slightly 250 ml/box (Bottle 1) (Bottle 1) swelling, no change 45 cfu/box23:05 Negative in milk appearance, (Bottle 2) (Bottle 2) pH decreased >0.5 Tetra Pack 1 day, 05:03 21:03 8 days, no swelling, 231 ml/box(Bottle 1) (Bottle 1) no change in milk 20 cfu/box 19:03 Negativeappearance, pH (Bottle 2) (Bottle 2) decreased to about 0.5 Prepack22:04 21:03 8 days, no swelling, 200 ml/box (Bottle 1) (Bottle 1) someprecipitates 44 cfu/box 24:04 Negative in milk, pH (Bottle 2) (Bottle 2)decreased > 0.5

This experiment showed that false negatives were obtained in the bottleswith media and only ten milliliters (mls) of sample. The false negativeswere not consistently obtained, so at least two bottles would berequired for bottles that contain only ten mls of sample combined withmedia. The two bottles are necessary to flag false positive or falsenegatives. Such false positives or false negatives would be flagged byinconsistent results for otherwise identical samples. False negativesdid not result when the sample size was 50 mls. The no-media testresults matched the reference method (both showed contamination), whilehalf of the bottles using media (with 10 ml sample size) generated falsenegative results.

TABLE 3 Effect of media on growth of organisms that could not grow inthe sample without the media for samples spiked with spiked with C.perfringens Lytic/ Anaerobic bottle Saline bottle Cat# 442265 Withoutmedia With media 50 ml/sample 10 ml/sample Time to Result Time to Result(h/min) (h/min) 36° C. Incubation Tetra Pack Negative 10:37 10 days, noswelling, 231 ml/box (Bottle 1) (Bottle 1) no change in milk 8 cfu/boxNegative Negative ) appearance, no (Bottle 2) (Bottle 2 change in pH, nocolonies on plates Prepack Negative 9:48 9 days, no swelling, 200 ml/box(Bottle 1) (Bottle 1) no change in milk 61 cfu/box Negative 9:58appearance, no (Bottle 2) (Bottle 2) change in pH, no colonies on plates

C. perfringens does not grow in those two kinds of packaged UHT milk, asdemonstrated by the results using the reference protocol. However, usingbottles with anaerobic media disposed therein yielded positive resultsfor those samples. This experiment demonstrates that analysis of samplein bottles without media yields results that correlate better to thetest protocol. This is because there is no media present to facilitatemicroorganism growth. Thus, the present method and system will not yieldfalse positives whereas bottles with media can facilitate the growth ofmicroorganisms that otherwise would not grow in the sample.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

The invention claimed is:
 1. A system for testing milk forcontamination, the system comprising: an incubator; a sample containerconfigured for use in an incubator that monitors a sample consisting ofmilk disposed in the container; a sensor disposed in the container, thesensor configured to monitor at least one parameter of the milk as themilk is heated in the incubator, the parameter being a parameter thatchanges in response to microbial growth in the sample wherein the sensoris positioned in the container such that, when the sample is introducedinto the container, the sensor is in contact with the sample; the systemprogrammed with a predetermined value of the monitored parameter, thatpredetermined value being indicative of microbial growth in the milk; areceptacle in the incubator for receiving the sample; a detector formonitoring changes in the sensor during incubation of the milk in theincubator; wherein the milk in the container is not combined with addedculture media for microbial growth.
 2. The system of claim 1 wherein themonitored parameter is selected from the group consisting of carbondioxide concentration, oxygen concentration and pH.
 3. The system ofclaim 1 wherein the incubation temperature is about 30° C. to about 38°C.
 4. The system of claim 1 wherein the volume of the sample is at leastabout 50 ml.